Immune tolerance is maintained, in part, by a process of clonal elimination in the population of immature lymphocyte. In particular, there is a well defined population of thymocytes that expresses both CD4 and CD8 (termed double positives or DPs), and this immature subset of thymocytes specifically undergoes apoptosis when challenged with antigen. The goal of this project is to define the biochemical mechanisms governing these events. The process of apoptosis is driven by the activation of caspases and the physiology of the mitochondria. The principal investigator plans to examine models of negative selection in the presence of endogenous and exogenously added inhibitors of caspases to determine which aspects of negative selection can be inhibited and whether forbidden self-reactive mature T cells appear in the thymus. The caspases present in the thymus and those that are activated as well as those absolutely required for antigen-mediated thymic deletion will be identified. Identified caspases will be the targets for genetic manipulation through the creation of deficient strains of mice. The impact of caspases on negative selection will be further studied by combining caspase deficiencies with the overexpression of Bcl-2, a protein that can act as a docking molecule and an ion channel regulating membrane potential of the mitochondria. Caspases will be further analyzed by transfection into a thymic cell line that mimics some of the aspects of thymocyte selection and development. Cell death in lymphocytes can be initiated by receptors in the TNFR family, and these receptors are known to act through the cytoplasmic mediator, FADD. Using mice that are deficient in FADD activity, preliminary data indicate that at least one model of negative selection is defective. Experiments are proposed to relate this defect to known cell surface and cytoplasmic mediators that bind FADD, and further experiments will identify FADD-interacting molecules that may be specifically expressed in the thymus. The long-term goals of this project are to establish a biochemical pathway that is important in the process of initiating apoptosis as a manifestation of clonal deletion in the thymus.